Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • ACK has been clearly implicated in cancer

    2024-02-19

    ACK1 has been clearly implicated in cancer progression in recent years. A strong correlation between ACK1 gene copy number, protein level and activity have been demonstrated in tumors of different tissue types (Mahajan et al., 2007; van der Horst et al., 2005; Wang et al., 2006). Our western blot analysis of different cancer cell lines confirmed a correlation of ACK1 protein levels with cancer aggressiveness. ACK1's role in proliferation and migration are clearly demonstrated and supported by reduced MAPK signaling in ACK1-silenced cells. Evidently, transient silencing of ACK1 does not affect Akt phosphorylation suggesting that ACK1 does not influence cellular metabolism and survival which are largely controlled by Akt. However, we do observed a reduction in Akt phosphorylation after prolonged ACK1 silencing using shRNA. This is probably due to a feedback mechanism as well as cross talk between the MAPK and Akt pathway in cells. In our work, in addition to the ACK1 WT potential in tumorigenicity, we also identified an enhanced migratory phenotype of the ACK1 S985 N mutant and its significant role in EMT in the A498 kidney carcinoma cells. The latter result is consistent with the observations of van der Horst et al. (2005) in which ACK1 WT was overexpressed in HMEC cells. Unlike their model where only fibronectin was upregulated, we showed here that in ACK1-silenced A498 cells, there was a strong upregulation of E-cadherin, an epithelial marker and downregulation of both N-cadherin as well as fibronectin. These differences suggest that the enhanced oncogenic ability arose from the S985 N mutation of ACK1 in A498 cells. The data convincingly implicated the involvement of ACK 1 in EMT as well as the role of ACK1 S985 N in A498 renal cancer cells. Its has been shown recently that A498 conditioned media could enhance invasiveness of 786-O, a less aggressive form of renal cell line (Chuang et al., 2008). The authors had identified involvement of several cytokines including TNF-α, Interleukin-1β, Interleukin-6, hypoxia-inducible factor-α and matrix metalloproteinase-2, of which TNF-α plays an important role. It would be exciting to further investigate how ACK1 contributes to EMT and if this involves the regulation of TNF-α. In addition to EMT, our finding also demonstrated that the single amino SLIGKV-NH2 mutation at residue 985 contributed to ACK1 protein stability with enhanced EGFR binding but impaired receptor ubiquitination and downregulation. These data are well supported by others showing the importance of the UBA domain on ACK1 for EGFR downregulation (Shen et al., 2007) and especially a recent publication by Chan et al. (2009), who demonstrated that overexpression of ACK1 blocked EGFR ubiquitination. This inhibition is overcome by co-expression of the Nedd4-2 E3 ligase which rapidly ubiquitinates and degrades ACK1. Here, we showed that the mutation on residue 985 on ACK1, resulted in a loss of its ubiquitin binding property. Thus, ACK1 S985 N does not respond to E3 ligase degradation and remain bound to EGFR in the presence of EGF stimulation. The physical binding of the ACK1 mutant might serve to reduce the ability of c-cbl to ubiquitinate EGFR, resulting in continuous mitogenic signaling in the renal cancer cells. The intriguing question is whether the level of Nedd4-2 E3 ligase also plays an important role in cancer development. Further, given that the EGFR-ACK1 S985 N protein complex dissociates after prolonged EGF stimulation, is there a proteasome-independent protein degradation pathway at play that removed ACK1 from the EGFR? All these questions await further investigation. Nonetheless, our data provide an explanation on how cancer cells could gain their oncogenic phenotype through various mutations of ACK1. In view of the effects of S985 N on ACK1 stability, we also attempted to characterize the second mutation found in gastric cancer cell line. The A634 T mutation located on the Nedd4-2 E3 ligase binding domain was verified in KatoIII. Unlike A498 cells, ACK1 of both wild type and mutant proteins are translated. An in vitro ubiquitination assay demonstrated that, like S985 N, the A634 T mutant is unable to bind ubiquitin (data not shown), suggesting that this mutation in the E3 ligase binding domain could confer stability to the kinase and therefore might be deficient in EGFR downregulation. However, we could not further investigate its role in EGFR regulation as we could not obtain a viable stable clone expressing A634 T constructs after G418 selection. Based on our data on ACK1 S985 N, it is highly likely that A634 T would behave similarly.